Laterally-fed membrane chromatography device

ABSTRACT

A method of forming a frame around a membrane stack for a laterally-fed membrane chromatography device is provided. The method includes placing a membrane stack having one or more membrane layers on a bottom surface of body of a master mold, the body having opposed side walls and opposed end walls, the opposed side walls spaced apart by a distance greater than a length of the membrane stack, the opposed end walls spaced apart by a distance greater than a width of the membrane stack; placing a cap on the body of the master mold to enclose the membrane stack in the master mold, the cap having at least one opening for injecting a material into a space defined by the end walls of the master mold, the side walls of the master mold, end walls of the membrane stack side walls of the membrane stack, the bottom surface of the body and an inner surface of the cap; injecting the material into the space around the membrane stack; and curing the material to form a frame around the membrane stack.

TECHNICAL FIELD

The following relates to a membrane chromatography device and morespecifically to a laterally-fed membrane chromatography device.

BACKGROUND

Membrane chromatography is a relatively new purification technique whichinvolves the use of a stack of synthetic membranes as chromatographicmedia. Membrane chromatography is emerging as a fast and cost-effectivealternative to resin-based column chromatography.

One attractive feature of membrane chromatography is the speed ofseparation. The predominantly convection-based transport of targetbio-molecules to and from their binding sites on a membrane, as opposedto the largely diffusion-limited mass transport of these moleculeswithin the resin bed makes membrane chromatography significantly faster.Membrane chromatography could therefore be faster by more than one orderof magnitude, a factor which contributes towards higher productivity anddecrease in product degradation by proteolysis, denaturation andaggregation.

The predominance of convection-based transport of target bio-moleculesalso makes it easier to model membrane chromatography. Also, in membranechromatography, the efficiency of binding of even large solutes such asmonoclonal antibodies is relatively independent of the superficialvelocity. This offers significant flexibility in process design. Otheradvantages include lower buffer usage and pressure drops, and theabsence of problems such as channeling and fracturing of resin beds.Moreover, the disposable nature of membrane devices eliminates the needfor cleaning and validation steps, and thereby contributes towardpracticality and ease of use.

The efficiency of membrane chromatography is critically dependent on thefluid flow distribution within the membrane device. Membranechromatography devices are commonly available in two formats: a) stackeddiscs, and b) radial flow. Both types of devices suffer from poor flowdistribution which can lead to shallow breakthrough and consequentlypoor binding capacity utilization.

Existing stacked disc devices often resemble syringe-type micro-filtersthat are relatively easy to fabricate and are used for preliminaryprocess development work. Stacked disks typically have large radial toaxial dimension ratios. The feed enters at a location corresponding tothe center of the first disk, while the flow-through is collected fromthe center of the last membrane in the stack. Consequently, the centralregion of the stack gets saturated with solute much earlier than theperipheral regions leading to poor breakthrough binding capacities.Radial flow devices have complicated design, and are used forlarge-scale purification. They have large dead volumes on both feed andpermeate side, and a large central core for supporting the membrane, andtherefore extremely poor device volume utilization.

SUMMARY

In one aspect, a method of forming a frame around a membrane stack for alaterally-fed membrane chromatography device is provided. The methodincludes placing a membrane stack having one or more membrane layers ona bottom surface of body of a master mold, the body having opposed sidewalls and opposed end walls, the opposed side walls spaced apart by adistance greater than a length of the membrane stack, the opposed endwalls spaced apart by a distance greater than a width of the membranestack; placing a cap on the body of the master mold to enclose themembrane stack in the master mold, the cap having at least one openingfor injecting a material into a space defined by the end walls of themaster mold, the side walls of the master mold, end walls of themembrane stack side walls of the membrane stack, the bottom surface ofthe body and an inner surface of the cap; injecting the material intothe space around the membrane stack; and curing the material to form aframe around the membrane stack.

In some other embodiments, the method further includes removing the cappiece and the master mold from the membrane stack and frame.

In some other embodiments, the curing the material includes cooling thematerial below a curing temperature.

In some other embodiments, the material is a thermoplastic polymer thatis injected into the master mold as a liquid and hardens when cooled.

In some other embodiments, the curing the material exposing the materialto ultraviolet light.

In some other embodiments, the placing the cap on the body of the mastermold includes resting the inner surface of the cap on a top surface ofthe membrane stack.

In some other embodiments, the placing the cap on the body of the mastermold includes resting the inner surface of the cap on an abutment memberof the body to support the cap above a top surface of the membranestack.

In some other embodiments, the placing the membrane stack on the bottomsurface of the body includes coupling the membrane stack to the bottomsurface of the body.

In some other embodiments, the placing the membrane stack on the bottomsurface of the body includes placing the membrane stack betweenretention ridges of the body to couple the membrane stack to the body.

In some other embodiments, the placing the membrane stack on the bottomsurface of the master mold includes positioning the membrane stack onthe bottom surface so the end walls of the membrane stack are adjacentto the end walls of the body and the side walls of the membrane stackare adjacent to the side walls of the body.

In some other embodiments, during the injecting the material into thespace around the membrane stack, the material is contained within thespace around the membrane stack.

In some other embodiments, during the curing of the material, thematerial adheres to the side wall and end walls of the membrane stack toform a frame around the membrane stack.

In another aspect, a membrane stack and frame formed by a methodprovided herein is provided.

In another aspect, a laterally-fed membrane chromatography device havinga membrane stack and frame formed by a method provided herein isprovided.

Other features and advantages of the present application will becomeapparent from the following detailed description taken together with theaccompanying drawings. It should be understood, however, that thedetailed description and the specific examples, while indicatingpreferred embodiments of the application, are given by way ofillustration only, since various changes and modifications within thespirit and scope of the application will become apparent to thoseskilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings included herewith are for illustrating various examples ofarticles, methods, and apparatuses of the present specification. In thedrawings:

FIG. 1 is a schematic diagram showing an exploded perspective view ofone embodiment of a laterally-fed membrane chromatography device;

FIG. 2 is a schematic diagram showing a cross-sectional view of alaterally-fed membrane chromatography device illustrating flow of afluid through the device;

FIG. 3 is a perspective view of a membrane stack and a frame, accordingto one embodiment;

FIG. 4. is an exploded perspective view of a master mould having a capand a body, according to one embodiment;

FIGS. 5A to 5E are schematic diagrams showing steps of a method offorming a frame around a membrane stack for a laterally-fed membranechromatography device, according to one embodiment;

FIG. 6 is a schematic diagram showing an exploded perspective view of asecond embodiment of a laterally-fed membrane chromatography device;

FIG. 7 is a perspective view of the laterally-fed membranechromatography device of FIG. 3;

FIG. 8 is a schematic diagram showing an exploded perspective view of athird embodiment of a laterally-fed membrane chromatography device withrounded channels;

FIG. 9 is a schematic diagram showing a cross-sectional view of thelaterally-fed membrane chromatography device of FIG. 6 illustrating flowof a fluid through the device;

FIG. 10 is a schematic diagram showing an exploded perspective view of afourth embodiment of a laterally-fed membrane chromatography device;

FIG. 11 is a graph showing a rate of purification of mono-PEGylatedlysozyme using the laterally-fed membrane chromatography device of FIG.6;

FIG. 12 is a picture showing SDS-PAGE results for a 4-hour reaction and2 mg/mL total protein samples using the laterally-fed membranechromatography device of FIG. 6;

FIG. 13 is another graph showing purification of mono-PEGylated lysozymeusing the laterally-fed membrane chromatography device of FIG. 6;

FIG. 14 is a picture showing SDS-PAGE results for 10-hour reaction and10 mg/mL total protein samples using the laterally-fed membranechromatography device of FIG. 6;

FIG. 15 is a graph showing separation of EG2-hFc using the laterally-fedmembrane chromatography device of FIG. 6;

FIG. 16 is a graph showing aggregate removal of IgG1 from HEK cell lineusing the laterally-fed membrane chromatography device of FIG. 6;

FIG. 17 is a graph showing aggregate removal of IgG1 from HEK cell lineusing the laterally-fed membrane chromatography device of FIG. 6;

FIG. 18 is a graph showing HIMC analysis of the IgG1 aggregateseparation peaks for separation conducted using the laterally-fedmembrane chromatography device of FIG. 6;

FIG. 19 is a picture showing Native PAGE analysis of the IgG1 aggregateseparation peaks;

FIG. 20 is a graph showing HI-LFMC results obtained with campath-1Hsamples;

FIG. 21 is a graph showing effect of dead volume on aggregate analysisof monomer-rich campath-1H using the laterally-fed membranechromatography device of FIG. 6;

FIG. 22 is a graph showing HI-LFMC for using the laterally-fed membranechromatography device of FIG. 8;

FIG. 23 is a collection of snapshots of feed and permeate sides of astacked-disk and a laterally-fed membrane module obtained during dyetracer experiments at 0, 3, 6, 9, and 12 seconds.

FIG. 24 is a representation of grayscale intensity data obtained fromsnapshots shown in FIG. 21 using Image J (membrane: hydrophilized PVDF;pore size: 0.22 micron; feed sample: 10 times diluted McCormick red fooddye; volume injected: 250 μL; flow rate: 10 mL/min).

FIG. 25 is a graph showing flow though lysozyme peaks obtained atnon-binding condition with stacked-disk (thin line) and laterally-fed(thick line) membrane modules;

FIG. 26 is a graph showing breakthrough curves for adsorption of BSA onthe anion-exchange membrane obtained using stacked-disk (thin line) andlaterally-fed (thick line) membrane modules;

FIG. 27 is a graph showing BSA elution peaks and conductivity profilesobtained using stacked-disk (thin line and thin dashed linerespectively) and laterally-fed (thick line and thick dashed linerespectively) modules;

FIG. 28 is a graph showing BSA elution peaks and conductivity profilesobtained from pulse binding experiments carried out using stacked-disk(thin line and thin dashed line respectively) and laterally-fed (thickline and thick dashed line respectively) modules;

FIG. 29 is a graph showing salt tracer peaks obtained with theradial-flow (thin line) and the laterally-fed (thick line) membranechromatography devices (membrane: Sartobind S; membrane bed volume: 7mL; feed: 0.5 M NaCl; running buffer: 20 mM sodium phosphate, pH=7.0;flow rate: 10 mL/min; volume injected: 2 mL (A) and 5 mL (B))

FIG. 30 is a graph showing salt breakthrough curves obtained with theradial-flow (thin line) and laterally-fed (thick line) membranechromatography devices;

FIG. 31 is a graph showing lysozyme elution peaks obtained with theradial-flow (thin lines) and the laterally-fed (thick lines) membranechromatography devices;

FIG. 32 is a graph showing lysozyme elution peaks obtained with theradial-flow (thin lines) and the laterally-fed (thick lines) membranechromatography devices;

FIG. 33 is a graph showing single-protein peaks obtained with ovalbumin(thin line), conalbumin (medium line), and lysozyme (thick line) usingthe radial-flow device;

FIG. 34 is a graph showing multi-component separation peaks obtainedwith the radial-flow device using 40 mL (A), 60 mL (B) and 80 mL (C)linear gradients;

FIG. 35 is a graph showing multi-component separation peaks obtainedwith the laterally device using 20 mL (A), 30 mL (B) and 40 mL (C)linear gradients; and

FIG. 36 is a graph showing an SDS-PAGE analysis of samples obtained fromthe multi-component protein separation experiment carried out with thelaterally-fed device using 40 mL linear gradient elution.

DETAILED DESCRIPTION

Various apparatuses or processes will be described below to provide anexample of each claimed embodiment. No embodiment described below limitsany claimed embodiment and any claimed embodiment may cover processes ormaterials that differ from those described below. The claimedembodiments are not limited to materials or processes having all of thefeatures of any one material or process described below or to featurescommon to multiple or all of the materials described below. It ispossible that a material or process described below is not covered byany of the claimed embodiments. Any embodiment disclosed below that isnot claimed in this document may be the subject matter of anotherprotective instrument, for example, a continuing patent application, andthe applicants, inventors or owners do not intend to abandon, disclaimor dedicate to the public any such embodiment by its disclosure in thisdocument.

It will be understood that the terms “top” and “bottom” referred toherein are used in the context of the attached Figures. The terms arenot necessarily reflective of the orientation of the laterally-fedmembrane chromatography device in actual use and are therefore not meantto be limiting in their use herein.

Described herein are various embodiments for a laterally-fed membranechromatography device that provides for the removal of a solute from afluid. The device houses a stack of flat sheet adsorptive membranes.Fluid enters the device at an inlet positioned at a first end of thedevice and is distributed laterally over a first side of a membranestack positioned at the first end of the device. The fluid then entersthe membrane stack at different locations along its length and flowsthrough the membrane stack in a direction normal to a top surface of atop membrane of the membrane stack. The fluid emerges from the membranestack at a bottom surface of a bottom membrane of the membrane stack andflows laterally with respect to the bottom surface of the bottommembrane of the membrane stack over a second side of the membrane stackuntil it is collected at the device outlet positioned at a second end ofthe device. The lateral-flows on both sides (e.g. the direction oftravel of the fluid over the first side and over the second side of themembrane stack) are parallel to each other.

Turning to the Figures, FIG. 1 shows one embodiment of a laterally fedmembrane chromatography device 100 according to one example. Themembrane chromatography device 100 comprises three bodies (e.g. plates):a top plate 102, a middle plate (e.g. frame) 104 and a bottom plate 106.

Top plate 102 is positioned superior to (e.g. on top of when device 100is in its normal configuration, as shown in FIG. 1) both middle plate104 and bottom plate 106. Top plate 102 defines an inlet 103 forreceiving a fluid at a first end 105 of device 100. In the embodimentshown in FIG. 1, inlet 103 is positioned at a first end 105 of device100 such that a fluid entering device 100 is received by inlet 103 andtravels through inlet 103 in a direction transverse to a direction offlow of the fluid through the membrane.

In the example shown in FIG. 1, a fluid can be provided to the membranechromatography device 100 through an inlet 103. Inlet 103 can be a portand is in fluid communication with first (e.g. top) channel 108 suchthat fluid entering the membrane chromatography device 100 is directedtowards membrane stack 109 via first channel 108. Further, inlet 103extends from an outer surface of device 100 through top plate 102 todirect fluid from a position outside of the device 100 towards membranestack of chromatography device 100.

As a fluid enters inlet 103 it is carried towards a leading edge 111 ofmembrane stack 109 (not shown) by top channel 108. Top channel 108(shown in FIG. 1 as a dotted line) is fluidly connected to inlet 103 andcarries fluid from inlet 103 towards membrane stack 109 (not shown). Assuch, inlet 103 is upstream of top channel 108 and top channel 108 isupstream of membrane stack 109. The term upstream can be defined asdirection of fluid flow experienced by (i.e. away from) a position on aflow pathway (i.e. channel or through membrane stack) relative to thedirection experienced by (i.e. towards) another position on the sameflow pathway (i.e. channel or through membrane stack). For example, alocation A of a flow pathway (e.g. top channel 108) is consideredupstream of a relative location B of the same flow pathway if, atlocation A, fluid is flowing away from location A and towards locationB.

Accordingly, the term downstream can be defined as direction of fluidflow experienced by (i.e. towards) a position on a flow pathway (i.e.channel or through membrane stack) relative to the direction experiencedby (i.e. away from) another position on the same flow pathway (i.e.channel or through membrane stack). For example, a location A of a flowpathway (e.g. top channel 108) is considered downstream of a relativelocation B of the same flow pathway if, at location A, fluid is flowingtowards location A from location B.

In the embodiment shown in FIG. 1, top channel 108 is defined by topplate 102 (e.g. is embedded in top plate 102 and formed by top plate102). It should be noted that in the embodiment shown in FIG. 1, inlet103 is positioned such that fluid entering device 100 via inlet 103 iscarried by top channel 108 towards membrane stack 109 (not shown) in adirection that is transverse to a direction of flow of the fluid throughmembrane stack 109. Put another way, in the embodiment shown in FIG. 1,as a fluid is received by inlet 103, the fluid has a direction of flowthat is lateral to (e.g. across) a direction of flow of the fluidthrough the membrane of device 100.

Top plate 102 can optionally have a vent 116 to vent the device 100(e.g. remove air bubbles therein) when not in normal operation. Topplate 102 can also optionally have a plurality of apertures 120 forsecuring top plate 102 to middle plate 104 and bottom plate 106. Topplate 102 can be secured to middle plate 104 and bottom plate 106 in anyappropriate manner (e.g. screws, bolts, pins, adhesives, etc.).

Middle plate 104 is positioned between top plate 102 and bottom plate106 (e.g. middle plate is positioned inferior (e.g. below). Middle plate104 is downstream of top plate 102 and upstream of bottom plate 106.Middle plate 104 defines space 113 for holding (e.g. housing) membranestack 109 (not shown).

Middle plate 104 has an inner wall 105 defining a cavity 113 for holdinga membrane stack 109. Membrane stack 109 has a leading edge 111 and atrailing edge 112. Leading edge 111 of membrane stack 109 is an edge ofa top surface of a top membrane of membrane stack 109 that receives thefluid from channel 108 (e.g. is fluidly coupled to channel 108) at firstend 105 of device 100. Trailing edge 112 of membrane stack 109 is anedge of a bottom surface of a bottom membrane of membrane stack 109 thatdistributes the fluid from the membrane stack 109 to second (e.g.bottom) channel 118 (e.g. is fluidly coupled to channel 118) at secondend 107 of device 100.

As fluid travels along channel 108 and approaches membrane stack 109,the fluid exits channel 108 and is distributed laterally over a topsurface (see for example top surface 225 of device 200 of FIG. 2) of atop membrane of the membrane stack 109. The direction of flow of thefluid changes as it enters (e.g. falls through) the membrane stack 109to a direction of flow that is transverse (e.g. orthogonal) to thedirection of flow of fluid along channel 108. The fluid enters membranestack 109 at different locations along a length and a width of membranestack 109 (e.g. at different locations along a length and a width of thetop surface of the top membrane of membrane stack 109). The direction offlow of the fluid changes again as it exits the membrane stack 109 to adirection of flow that is transverse (e.g. orthogonal) to the directionof flow of fluid through the membrane stack 109. The fluid entersmembrane stack 109 at different locations along a length and a width ofmembrane stack 109 (e.g. at different locations along a length and awidth of the top surface of the top membrane of membrane stack 109) andexits membrane stack 109 at different locations along a length and awidth of membrane stack 109 (e.g. at different locations along a lengthand a width of the bottom surface of the bottom membrane of membranestack 109). An exemplary flow path of the fluid through membrane stack109 is shown in FIG. 2. In one example, the flow of the fluid throughmembrane stack 109 is in a direction normal to a plane defined by thetop surface of the top membrane of the membrane stack 109. In oneexample, the middle plate 104 can be made of Delrin® (Dupont).

Membrane stack 109 can comprise one or more membrane sheets. The device100 (see FIG. 2) is designed to house a stack of rectangular flat sheetadsorptive membranes 109. Each membrane sheet of stack 109 can be anyflat sheet adsorptive membrane appropriate for chromatographyapplications. For example, the membrane stack 109 may be appropriate forprotein separation. Membrane stack 109 can be incorporated into device100 in any appropriate manner. In one specific example, standard PEEKfittings can be employed to integrate the membrane chromatography devicewith an AKTA Prime liquid chromatography system (GE HealthcareBioscience, QC, Canada). In one example, the membrane stack comprises atleast one hydrophilized poly(vinylidene fluoride) (PVDF) membrane. Also,membrane stack 109 can have a thickness greater than a thickness ofmiddle plate 104 to provide that the device 100 is sealed when theplates 102, 104, 106 are sandwiched together (e.g. when device 100 isassembled).

In some embodiments, the membrane stack (e.g. membrane stack 109) can beassembled into a module (for example but not limited to middle plate104) for use in a laterally-fed membrane chromatography device byforming a frame around the membrane stack. A perspective view of oneexemplary module 250 having a membrane stack 252 and a frame (or gasket)254 is shown in FIG. 3.

Membrane stack 252, as described previously, can be held by a frame 254that substantially surrounds side and/or end surfaces of the membranestack 252. In the embodiments shown in the Figures, side surfaces 256,258 and end surfaces 260, 262 of membrane stack 252 are surrounded byframe 254 and the top surface of the top membrane and the bottom surfaceof the bottom membrane remain open to receive and provide, respectively,the fluid filtered by the membrane stack 252. The person skilled in theart will understand that membrane stack 252 may have any desired shape(for example but not limited to a circular shape, a square shape, arectangular shape, etc.) and frame 254 can be configured to surround anyside and/or end surfaces of the membrane stack while the top surface ofthe top membrane and the bottom surface of the bottom membrane remainopen to receive and provide, respectively, the fluid filtered by themembrane stack 252.

Module 250 is configured so that the fluid is directed (for example butnot limited to from top plate 102) through the membrane stack 252 in afluid path that is substantially perpendicular to a plane defined by thetop surface of the top membrane of the membrane stack 252 and/or to aplane defined by the bottom surface of the bottom membrane of themembrane stack 252. Frame 254 of module 250 is configured to contain orcarry the membrane stack 252 and to constrain the flow of the fluid towithin the membrane stack 252 such that all, or a desired portion, ofthe fluid flow passes through the membrane stack 252 and does not escapearound the membrane stack 252. Accordingly, frame 254 may besubstantially impermeable to the fluid.

Membrane stack 252 may be integrally molded such that the membranesheets of the membrane stack 252 are a single unit. In one example,membrane stack 252 may be of the form of a flat disk. As discussedabove, membrane stack 252 can be joined to a feed/distributor systemsuch as but not limited to top plate 102 and bottom plate 106 to form amembrane chromatography device 100. As shown in FIG. 3, membrane stack252 has a length L1 and a width W1.

Frame 254 may be made of a polymeric material that is injectable intothe space 408 as a liquid and then curable into a solid. For example,frame 254 may be made of a thermoplastic polymer that is liquid whenwarm and cools to form a solid material. For example, frame 254 extendsform the outer side sand end surfaces of the membrane stack

To form a frame 254 around membrane stack 252, a master mold may beused. As shown in FIG. 4, a master mold 270 may have two parts: a body272 for housing the membrane stack 252 and cap 274 for sealing themembrane stack 252 in the body 272. Body 272 has opposed inner endsurfaces 280 and 282 spaced apart by a distance L2 and opposed innerside surfaces 284 and 286 spaced apart by a distance W2.

Cap 274 has at least one opening 294 for receiving the polymericmaterial into the enclosed space 278 for forming the frame 257 aroundthe membrane stack 252. Cap 274 may have a second opening 295 to act asa vent when opening 294 is receiving the polymeric material into theenclosed space 278 for forming the frame 254.

Cap 274 may couple to body 272 in any manner known in the art. In theembodiment shown in FIG. 4, cap 274 has a length L3 and width W3 that isthe same as length L2 and width W2 of body 272 such that cap 274 mayinsert into a top portion of body 272 to sealingly engage body 272 andseal membrane stack 252 within body 272.

As length L2 and width W2 of body 272 are greater than length L1 andwidth W1 of membrane stack 252, when membrane stack 252 is placed inbody 272 and cap 274 is placed on top of body 272, inner end surfaces280 and 282 and inner side surfaces 284 and 286 of body 272, bottomsurface 288 of body 272, and inner surface 290 of cap 274 co-operatewith membrane stack 252 to provide an enclosed space 278 surrounding theside surfaces 256, 258 and the end surfaces 260, 262 of the membranestack 252. The skilled person will understand that the inner walls 280,282, 284, 286 of body 272, bottom surface 288 of body 272, and innersurface 290 of cap 274 may have any size or shape to provide a framehaving any size or shape surrounding the side walls and/or end walls ofmembrane stack 252. The height of the enclosed space 278 is generallyequal to a height H of the membrane sack.

In some embodiments, membrane stack 252 can be coupled to bottom surface288 prior to placing cap 274 on body 272 to retain membrane stacktherein. For instance, membrane stack 252 can be coupled to bottomsurface 288 prior to placing cap 274 on body 272 by applying weight onthe top mold. In this manner, the membrane stack may be coupled with thebottom surface of the bottom mold and the polymer may not affect thesurface of the membrane stack on the bottom side.

In other embodiments, bottom surface 288 may include retention ridgessuch as retention ridges 297 approximately sized to fit the membranestack 252 to retain the membrane stack 252 in a central position onbottom surface 288 when the space 278 is filled with the polymericmaterial. In still other embodiments, membrane stack 252 can be held inplace by cap 274 contacting and directing a downward force upon membranestack 252 when cap 274 is positioned on top of body 272.

FIG. 5 shows a method 500 of forming a membrane stack of flat sheetmembranes.

Method 299 includes a first step 299A of placing membrane stack 252 onto bottom surface of master mold 270. As described above, membrane 252may be coupled to bottom surface 288 using an adhesive, retention ridges297, or held in place by a downward force of cap 274.

At step 299B, cap 274 is placed onto body 272 to form space 278. Cap 274sealingly engages body 272 to seal membrane stack 252 within the body272 and to form a space 278 for injecting a polymeric material in tospace 278. In some embodiments, bottom surface 290 of cap 274 will restagainst a top surface of a top membrane of membrane stack 252 when cap274 sealingly engages body 272. In other embodiments, bottom surface 290of cap 274 may be spaced from a top surface of a top membrane ofmembrane stack 252 when cap 274 sealingly engages body 272. In thismanner, body 272 may have an abutment member 293 (see FIG. 4) thatengages bottom surface 290 when cap 274 seals body 272 to support cap274 above membrane stack 252.

At step 299C, the polymeric material is injected into the space 278through an opening 294 of the cap 274. The polymeric material may flowvia gravity through the opening 294 into the space 278 surrounding themembrane stack 252. The polymeric material does not substantially flowlaterally into the membrane stack 252 when injected into the space 278.

At step 299D, the polymeric material cures to form frame 274 aroundmembrane stack 252. In some embodiments, the polymeric material may be athermoplastic polymer material and cure by cooling to a temperaturebelow a curing temperature of the material. In other embodiments, othermechanisms may be used to cure the polymeric material. For instance, thepolymeric material may comprise a photoinitiator and cure upon exposureto ultraviolet (UV) light. In one example, the polymeric material may bea polyurethane based polymer. In some embodiments the curing times canvary from tens of minutes to overnight (e.g. in a range of about 8 to 12hours).

At step 299E, the cap 274 and the body 272 of the master mold 270 areremoved from the membrane stack 252 and frame 274. Upon removing the cap274 and the body 272 the membrane stack 252 and frame 274 can beincorporated into a membrane chromatography device such as the laterallyfed membrane chromatography device 100.

The above method 299 may be used to fabricate a module 270 such as butnot limited to the middle plate 104 of the laterally-fed membranechromatography (LFMC) device using polyurethane as the framing polymermaterial. Silicon rubber was used to make the master mold, i.e. both thetop and bottom parts. The silicon hardener was mixed with the base in a1:10 ratio to fill the blank which was designed using Autodesk Inventor.Membrane stacks of different thicknesses were prepared using thisapproach (an example is shown in FIG. 10).

As described above, a fluid is laterally distributed over the topsurface (e.g. feed-side) of the membrane stack 109 and thereby enters(e.g. passes through) the membrane stack 109 at different locationsalong its length, eventually emerging at corresponding locations of thebottom surface (e.g. on the permeate side), where the fluid flowslaterally to the outlet 123 of the device 100. This configuration makesit possible to balance the pressure-drop on the feed side with that onthe permeate side, thereby ensuring uniformity of flow along the lengthof the membrane stack 109. Also, unlike a radial-flow device, where thesuperficial velocity within the bed increases in a radially inwarddirection, the flow of fluid through device 100 can be more uniform(e.g. fluid passing through a greater proportion of the membrane stack109 when compared to radial-flow prior art devices). As shown in FIG. 2,the fluid has a flow path length that is independent of where the fluidenters the membrane stack 109. Therefore, the device 100 can provide forfluid passing therethrough to have a consistent path length through thedevice 100. This may improve the efficiency of membrane utilization andthe resolution of eluted peaks in chromatographic separation.

In one example, top channel 108 extends from inlet 103 in a directionlateral to middle plate 104 (and therefore also membrane stack 109) andbottom channel 118 extends towards outlet 123 in a direction lateral tomiddle plate 104 (and therefore also membrane stack 109). Put anotherway, channels 108 and 118 can extend towards membrane stack 109 toprovide that inlet 103 and outlet 123, respectively, are offset frommembrane stack 109.

In one example, top channel 108 and bottom channel 118 can have anirregular shape (see FIGS. 6 and 7). In one example, as top channel 108extends laterally from inlet 103 to leading edge 111, a width of topchannel 108 can increase over the length of top channel 108. Forexample, a width of the top channel 108 can increase at a constant rateover its length (e.g. taper) or at a variable rate over its length (e.g.rounded). An irregular (e.g. tapered or rounded) shape of top channel108 may provide for distribution of the fluid over the leading edge 111of membrane stack 109 as fluid is provided by channel 108 from inlet 103to membrane stack 109.

Similarly, in one example, as bottom channel 118 extends laterally fromtrailing edge 112 to outlet 123, a width of bottom channel 118 candecrease over the length of bottom channel 118. For example, a width ofthe bottom channel 118 can decrease at a constant rate over its length(e.g. taper) or at a variable rate over its length (e.g. rounded). Anirregular (e.g. tapered or rounded) shape of bottom channel 118 mayprovide for collection of the fluid from trailing edge 112 of membranestack 109 as fluid is provided by channel 118 from membrane stack 109 tooutlet 123.

In some examples, top channel 108 and bottom channel 118 can each,independently, have a structure therein to disrupt the flow of fluidthere through. For example, top channel 108 and bottom channel 118 cancomprise a mesh layer (e.g. a structure having a pattern to disrupt flowwithin the channels 108,118). In some examples, the mesh layer 122 (notshown) within top channel 108 and bottom channel 118 can have a samethickness as top channel 108 and bottom channel 118. In another example(see FIG. 6, below) a plurality of pillars (e.g. microcolumns) can beprovided within top channel 108 and bottom channel 118 to disrupt theflow of fluid there through. Structures (e.g. spacers) as describedherein positioned in channels 108,118 can be used to provide each ofchannels 108,118 with similar lateral resistance over the sides (e.g.top surface and bottom surface of membrane stack 109 and/or leading edge111 and training edge 112) of the membrane stack 109. The structures canalso provide support for membrane stack 109 and may reduce dead volumewithin device 100. In one example, before assembling the device 100,channels 108 and 118 may be provided with 70 mm×20 mm pieces of wovenwire mesh (approximately 0.5 mm thick)

Middle plate 104 as shown in FIG. 1 also defines a plurality ofapertures 121 extending therethrough for securing middle plate 104 totop plate 102 and bottom plate 106. As described above, top plate 102can be secured to middle plate 104 and bottom plate 106 in anyappropriate manner (e.g. screws, bolts, pins, adhesives, etc.). In theembodiment shown in FIG. 1, the middle plate 104 is glued to the topplate 102 and bottom plate 106, however middle plate 104 can be coupledto the top plate 102 and the bottom plate 106 in any appropriate manner.

The direction of flow through membrane stack 109 is analogous to thefluid flow pathway shown in FIG. 2, where FIG. 2 shows a schematic viewof a laterally-fed membrane chromatography device where inlet 203 andoutlet 223 of device 200 direct fluid to and from membrane stack 209,respectively, in a direction that is parallel to the direction of flowof fluid through membrane stack 209. In contrast, inlet 103 and outlet123 of device 100 direct fluid to and from membrane stack 109,respectively, in a direction that is transverse to the direction of flowof fluid through membrane stack 109. However, the direction of flow offluid along top surface 225 and bottom surface 227 of membrane stack 209as shown in FIG. 2 is analogous to the flow of fluid along the topsurface and the bottom surface 127 (e.g. and collecting surface 130) ofthe device 100 of FIG. 1.

The fluid passing though membrane stack 109 can emerges from a bottomsurface of a bottom membrane of the membrane stack 109 onto a collectingsurface 130 of bottom plate 106. Collecting surface 130 can be formedinto bottom plate 106 as shown in FIG. 1. Collecting surface 130 may beconfigured to direct fluid in a direction transverse to a direction offlow through the membrane stack 109 to bottom channel 118 for deliveringthe fluid to outlet 123 of device 100. The direction of flow of thefluid through channel 123 is transverse to the direction of flow offluid through the membrane stack 109 and can be the same direction asthe direction of fluid through channel 108. Device 105 also has a secondend 107 opposed to first end 105.

Bottom plate 106 as shown in FIG. 1 also defines a plurality ofapertures 122 extending therethrough for securing bottom plate 106 tomiddle plate 104 and top plate 102. As described above, top plate 102can be secured to middle plate 104 and bottom plate 106 in anyappropriate manner (e.g. screws, bolts, pins, adhesives, etc.).

In one example, top plate 102, middle plate 104 and bottom plate 106 canbe acrylic plates 3D printed using a commercially available 3D printer(e.g. ProJet HD3000 printer by 3D Systems (Rock Hill, S.C., USA)). Topplate 102, middle plate 104 and bottom plate 106 can also be formed bypolymer molding.

Plate 102 and 106 are generally made of polymer-based materials. In oneexample, each of the top plate 102 and the bottom plate 106 can be3D-printed with acrylic-based polymers.

As can be seen in FIG. 2, the flow path lengths independent of the pathtravelled by the fluid through the membrane are generally the same. Thismay improve efficiency of membrane utilization (e.g. solute removal fromthe fluid) and thereby provide higher breakthrough binding capacity forthe device.

FIG. 6 shows another embodiment of a laterally-fed membranechromatography device 300. In this embodiment, top plate 302 and bottomplate 306 define top channel 308 (not shown) and bottom channel 318 forcarrying fluid towards and away from, respectively, membrane stack 309(not shown). In this embodiment, top channel 308 and bottom channel 318are tapered channels (see 318) for distribution of the fluid over theleading edge 311 on the feed side and collection of the fluid of thefluid from the trailing edge 312 on the permeate side. For example, asshown in FIG. 6, bottom channel 318 is shown to reduce in width alongits length as it direct fluid from the collecting surface

In FIG. 6, pillars 355 are positioned in each of channels 308 and 318.The use of pillars 355 in channels 308 and 318 can provide for loweringlateral resistance within channels 308 and 318 and support membranestack 309. In one embodiment, reducing the dimensions (e.g. length,width and depth) of channels 308 and 318 can reduce the dead volume ofdevice 300.

As shown in FIG. 6, plates 302 and 306 are provided with vents 340 and341, respectively. Vents 340 and 341 can be used to prime the device 300and/or to remove of air bubbles trapped in the channels 308, 318,respectively.

FIG. 7 provides a picture of one example of a membrane chromatographydevice according to the embodiment described with respect to FIG. 6. Inthe example shown in FIG. 7, middle plate 304 was made of PVC.

FIG. 8 shows an exploded view of another embodiment of a laterally-fedmembrane chromatography device 500. In this embodiment, the membranestack 509 is not coupled (e.g. glued) within the middle plate 504.Rather, the width and length of membrane stack 509 is shown to besimilar to the width and length of the space 513 of middle plate 504.Also, the thickness of the membrane stack 509 is greater than thethickness of the middle plate 504 to provide that the device 509 issealed when the plates 502, 504, 506 are sandwiched together.

FIG. 9 is a cross-sectional diagram showing an exemplar flow pathwaythrough the membrane chromatography device of FIG. 6. In thisembodiment, inlet 303 and outlet 323 are shown as directing fluid intothe device 300 from a top surface 350 and out of the device 300 from abottom surface 351, respectively. In this example, inlet 303 and outlet323 are shown as directing fluid in a direction transverse to thedirection of fluid through the first channel 308 and second channel 318,respectively (e.g. transverse to the direction of fluid flow along a topsurface 307 and bottom surface 317 of membrane stack 309). In anotherexample, inlet 303 and outlet 323 can direct fluid to flow in adirection perpendicular to the direction of flow of fluid through firstchannel 308 and second channel 318, respectively. Further, in theexample shown in FIG. 9, inlet 303 and outlet 323 are positioned to beoffset from the membrane stack 309. It should be noted that inlet 303and outlet 323 can be positioned to be offset from the membrane stack309 (e.g. by a length of top channel 308 or bottom channel 318,respectively) or be positioned aligned with leading edge 311 andtrailing edge 312, respectively (e.g. a lateral distance between theinlet 303 and outlet 323 can match a length of membrane stack 209). Putanother way, the inlet 303 being offset from the membrane stack 309refers to the inlet 303 being laterally spaced from or distanced fromleading edge 311 of the membrane stack 309 such that fluid entering thedevice 300 through the inlet 303 travels through the top channel 308 ina direction lateral to the direction of flow of the fluid through themembrane stack 309 to pass from the inlet 303 to the leading edge 311(e.g. and the top surface 307) of the membrane stack 309. Likewise theoutlet 323 being offset from the membrane stack 309 refers to the outlet303 being laterally spaced from or distanced from the membrane stack 309such that fluid exiting the device 300 through the outlet 323 travelsthrough the bottom channel 318 in a direction lateral to the directionof flow of the fluid through the membrane stack 309 to travel from thetrailing edge 312 (e.g. and the bottom surface 317) of the membranestack 309 to the outlet 323. It should be noted that in one embodiment,top channel 308 and bottom channel 318 can have a same length to providefor equal pressure distribution across membrane stack 309.

In the embodiment shown in FIG. 9, the inlet 303 extends form topsurface 350 of device 300 to top channel 308 to receive fluid into thedevice 100 and direct the fluid to top channel 308. Outlet 323 extendsfrom bottom channel 318 to collect fluid from bottom channel 318 anddirect the fluid out of the device 300.

In one example, the membrane chromatography devices described herein canbe stacked to provide multiplexed systems for complex separations ofmultiple solutes from the fluid.

EXAMPLES

A device according to the embodiment shown in FIG. 6 was used toretrieve the first set of example results provided below. The device wasfabricated with two different bed heights which gave the membrane bedvolumes of 1 mL, 4.6 mL, and 9.2 mL as opposed to the 7 mL for theinitial device described in.

Finally, cation exchange S membranes were glued in the frame using epoxyglue and the three layers were assembled using Weldon#16 adhesive. Thefinal 9.2 mL device is shown in FIG. 7. Luer fittings were used toconnect the inlet and outlet to the AKTA prime (GE health-carebiosciences). Two other terminals were used for removing the bubbleswhich were blocked during the device operation.

The details of the device design, including the dimensions, membrane bedheight, and bed volumes for the so far developed LFMC devices areavailable in Table 1.

TABLE 1 Design details of the LFMC devices Membrane Number Outer bed Bedof Membrane dimension volume height membrane dimensions Pillar of plate(mL) (mm) layers (mm × mm) array (mm x mm) 9.2 6.6 24 70 × 20 28 × 7 150× 40 4.6 3.3 12 70 × 20 28 × 7 150 × 40 1.0 2.7 10 38 × 10 15 × 3 120 ×30

Analytical Hydrophobic Interaction Membrane Chromatography (HIMC)

The laterally fed membrane chromatography device as described above withreference to FIG. 7 was tested using a stack of rectangularhydrophilized poly(vinylidene fluoride) (PVDF) membranes. The top andbottom plates contained rectangular channels with dimensions of 100mm×10 mm with curved ends. The height of the channel was 0.2 mm. Theinlet and outlet port were both perpendicular to the plates. Thechannels contained hexagonal arrays of pillars for better flowdistribution in the lateral direction. The membrane stacks were placedwithin the slot of a spacer with the thickness slightly lower than themembrane stack. The dimension of the sloth was 1.5 mm bigger than therectangular curved channels from each side. The layers were sandwichedtogether using screws over the length of the device. The blowout diagramof the device is shown in FIG. 8.

Purification of PEGylated Proteins

PEGylation is one of the major post-translational modifications oftherapeutic proteins in which polyethylene-glycol, a hydrophilic andinert polymer, is covalently attached to protein molecules. The increasein the hydrodynamic diameter of the molecule reduces the renal clearancerate; therefore, prolonging the circulation half-life of the proteindrug and diminishing the number of needed drug administration. Moreover,PEG molecules shield the surface of the proteins which results indecreased proteolysis and aggregation as well as higher solubility.Protein PEGylation is usually carried out in a liquid batch reaction.The reaction products include mono-PEGylated proteins, di-PEGylatedproteins, and higher PEGylated forms as well as the unreacted proteinand PEG molecules. However, only the mono-PEGylated protein is thedesired product and therefore it needs to be purified from othermoieties available in the reaction mixture. The approaches for theseparation of PEGylated proteins have been majorly based on charge,size, and hydrophobicity difference of the unreacted reagents anddifferent PEGylated proteins. However, the most famous technique forpurification of mono-PEGylated protein is the ion-exchange separationmajorly using cation-exchange media. The fractionation is based on theinteraction of PEGylated proteins with the cation-exchange media whichgoes weaker as the extent of PEGylation is increased. This issignificantly owing to the shielding of the surface charges of theprotein molecule by the neutral PEGs. Therefore, when the salt gradientis used for elution, the higher PEGylated proteins elute faster,followed by the mono-PEGylated form and the native proteins. It isnoteworthy that the differences between the adsorption binding strengthfor different PEGylated proteins are very subtle which makes theseparation very challenging.

Purification of PEGylated lysozyme was performed using the 9.24 mLlaterally-fed cation-exchange membrane chromatography device. PEGylationreactions were carried out using 5 kDa PEG and lysozyme as the modelprotein following the aldehyde chemistry. The reaction was carried outin 15 mL vials for 4 hours and then desalted by 3 kDa MWCO centrifugalultra-filters. The retentate was diluted to acquire a certain totalprotein concentration which was then injected to the LFMC device. Theinjection was followed by a gradient to the eluting buffer containing0.5 M sodium chloride. Subsequently, the gradient was optimized so thatto give high-resolution of separation within the shortest volume. Theresults from the 4-hour reactions with 2 mg/mL total proteinconcentration are shown in FIG. 11.

The optimized elution conditions was achieved when the sample wasinjected at 20% eluting buffer followed by a linear gradient to elutethe di- and mono-PEGylated proteins, and a step-change elution to obtainthe unreacted lysozyme. Different PEGylated proteins were fractionatedwithin 10 membrane bed volumes (MBV=9.24 mL) and the peaks werecollected and further analyzed by gel-electrophoresis. The results fromthe SDS-PAGE are shown in FIG. 12. Although separation was carried outat 15 mL/min (˜1.5 MV/min) flow rate the resolution of separation isvery high.

The results obtained from the separation of mono-PEGylated lysozyme werecompared with the preparative techniques available in the literatureusing the packed-bed columns with the same sulphonated (S) strong cationexchange ligands. The reaction residence time as well as the PEGmolecular size was consistent with the experimental conditions used forthe LFMC device. GigaCap S-650 resins was used as the chromatographymedia having 13.4 mL bed volume and the separation was carried out at 1mL/min. The results confirmed that the LFMC device gave comparableresolution of separation with the packed-bed columns. Plus, theseparation was carried out with much greater speeds which is highlybeneficial in large-scale manufacturing.

Separation of Monoclonal Antibody (mAb) Aggregates

Monoclonal antibodies (mAbs) are considered as the most prevalenttherapeutic proteins in the biopharmaceutical industry. Manufactured byrecombinant technology, mAbs have high tendency to self-associate intoaggregates as a cause of high concentration as well as extreme shearrates and pH conditions in different stages of the manufacturingprocess. Antibody aggregation is highly unwanted as it usually leads toincreased immunogenicity, loss of biological activity and decreasedsolubility of the therapeutic protein. Therefore, antibody oligomersshould be separated from the final product. Separation during thedownstream processing is the biggest opportunity to polish the antibodyaggregates. Size-based analysis and separation of mAbs aggregates isvery slow and often gives poor resolutions. This is while ion exchangechromatography has shown to be very useful in production scale polishingof aggregates. In the most recent strategies, cation exchangechromatography (CEX) is employed as the intermediate purification stepin the downstream processing of mAbs which is taken place after themajor protein capture step and is followed by anion-exchange polishingsteps. CEX is carried out in the bind-and-elute mode in which impuritiessuch as host cell proteins (HCPs), DNA, antibody fragments and solubleantibody aggregates are majorly separated from the target protein. Morespecifically, dextran-grafting cation exchangers have been widelyexploited in process scale bioseparation. In the pH conditions that arenot the pI of protein, protein aggregates bind more strongly to thecation exchangers.

Hydrophobic interaction membrane chromatography (HIMC) has beeninvestigated for analysis and separation of protein aggregates. Incomparison with CEX columns, the application of adsorptive membranes iscost-effective and the technique brings about rapid processing due tothe convective solute transport. This is while samples have to beprepared in high concentration of anti-chaotropic salt conditions.

We investigated the performance of the LFMC device in the preparativescale (4.62 mL cation exchange membrane) for separation of antibodyaggregates. Initially, purification of heavy chain monoclonal antibodyEG2-hFc was taken into consideration. EG2-hFc is a camelid chimericheavy chain mAb, genetically engineered to have human Fc region.Therefore, protein-A chromatography was used as a capture step forsamples from the day 7 of cell culture. However, protein-A columns arenot capable of removing any mAb aggregates due to the fact that Fcregion does not get affected in the aggregation process.

Preliminary experiments were run to find the optimized pH for theseparation of EG2-hFc aggregates. The values of 5.0, 5.5, and 6.0 werestudied and pH=6.0 gave the best results. Protein-A purified EG2-hFcsamples having concentration of 0.05 mg/mL and volume of 5 mL wereinjected in the device. With the linear gradient elution of 30 mL to theeluting buffer which contained 0.5M sodium chloride, the EG2-hFcaggregates were resolved. Higher gradient (60 mL) was also examined fornear baseline separation (FIG. 15). It is noteworthy that although theseparations were run at 20 mL/min flow rates the resolution ofseparation was high enough to resolve the aggregates within only fivemembrane bed volumes. The LFMC device offers high resolution and highthroughput separation of mAb aggregates with cation exchange mechanism.

The separation of IgG1 aggregates from the HEK 293 cell line kindlydonated from Durocher lab at National Research Council of Canada,Montreal was also investigated. Samples having 0.5 mg/mL total proteinand 2 mL volume were injected to the LFMC device. Other experimentalconditions were kept the same with the EG2-hFc runs described above.Initially, different linear gradients to the eluting buffer within therange of 100 mL and 300 mL were examined (FIG. 16). The 300 mL gradientgave the best separation of aggregates which is shown in FIG. 17.

Higher sample concentrations and volumes were experimented to challengethe LFMC device. The flow through peak as well as the three elutingpeaks was collected and analyzed using hydrophobic interaction membranechromatography (HIMC) technique. The feed concentration was set to 0.2mg/mL and the collected peaks were concentrated accordingly (FIG. 18).The peaks were also analyzed using 7.5% Native PAGE, the results ofwhich are shown in FIG. 19. The results confirm the separation of themonomeric IgG1 from the aggregates when relatively high concentrationsof the mAbs were processed. The LFMC device is capable of resolving themonomeric IgG1 mAbs from the aggregated forms within 20 membrane bedvolumes. It is noteworthy that compared to the EG2-hFc experiments, thesamples have high aggregate contents which makes the separation highlychallenging.

The LFMC device offers high resolution purification of mAbs in thebind-and-elute mode with high throughputs. Considering the easy scale-upof the device, the LFMC device has great potentials to serve as theintermediate separation stage in the production of mAbs, helping withdecreasing the processing time, buffer usage, and moving towardsdisposable chromatography units.

Ultra-Fast Analysis of Monoclonal Antibody (mAb) Aggregates

The current state of the art for rapid analysis of mAb aggregates issize exclusion chromatography based on the use of sub-2 micro resinparticles by ultra-high pressure liquid chromatography (UPLC). Makinguse of such small resin particles diminished the flow path length andprovides higher number of theoretical plates per unit length of thecolumn. Therefore, the UPLC columns have shorter volumes and are runwith typical flow rates due to which they provide much higher throughputcompared to conventional HPLC systems. However, the fast assay time areat the sacrifice of the pressure. UPLC systems are operate at one orderof magnitude higher pressures which requires costly pumps and sealingequipment.

The analytical device described in FIG. 8 was used for ultra-fastseparation of mAb aggregates. Three layers of PVDF membrane were housedwithin the device providing the bed volume of 0.4 mL. The separation wasbased on the higher binding strength of the aggregates compared to thenative monomeric antibody. The experiments were carried out using linearnegative salt gradients at 16 mL/min. As a result of high operating flowrate, the aggregate analysis was achieved in less than 1.5 min. FIG. 20demonstrates the results obtained with Chinese hamster ovary (CHO)derived Alemtuzumab (campath-1H). Chromatogram A showing the aggregateanalysis for an aggregate-rich sample whereas B was obtained with amonomer-rich sample. High-resolution monomer/aggregate separation wasattained in very short assay time.

In comparison, due to low bed height of the membrane stack, the pressuredrops were below 200 kPa. Comparing the hydrophobic interactionlaterally-fed membrane chromatography (HI-LFMC) discussed with the UPLCtechniques for mAb aggregate analysis, the HI-LFMC is extensivelycheaper, the pressure drops are much lower, and the devices can be usedin a single-use manner. The possibility of having such an ultra-fastassay which can be run with bench-top liquid chromatography machinessuch as AKTA systems is highly advantageous.

The dead volume of the analytical device was further decreased byincreasing the pillar size in both the feed and the permeate channel.The device was tested with campath-1H monomer-rich sample shown in FIG.21. While chromatogram C is the repeat for the results shown in FIG. 20,B, FIG. 21, B illustrates the chromatogram obtained with the device withlower dead volume. Evidently, the peaks obtained are much sharper.Therefore, the separation was also carried out with 30 mL lineargradient shown in FIG. 20, A which helped with decreasing the separationtime even further. The validity of the technique was then approved fortwo other mAb samples, HIgG1-CD4 (campath-9) and human embryonic kidney293 (HEK) cell derived IgG1 (Trastuzumab biosimilar).

Module Design: Comparison with Stacked-Disk Devices

The performance of a laterally-fed membrane device according to FIG. 10was compared with that of an equivalent centrally-fed, disc-basedmembrane module using a single layer membrane. Anion-exchange membranesheets having the same surface area and thickness, and thereby same bedvolume were used in both devices. Tracer experiments were carried outusing a dye as well as a protein (lysozyme) under non-binding condition.Bovine serum albumin (or BSA) was used as model binding protein todetermine the binding capacities of membrane sheets of identical surfacearea and bed volume housed in the and stacked-disk modules were comparedin the breakthrough and pulse modes. The results obtained are discussed.

The circular module had an outer diameter of 75 mm while the laterallyfed module had an external dimension of 200 mm×40 mm. Membraneassemblies consisting of the adsorptive membrane sandwiched between twoplastic shim layers as spacers (each of 0.3175 mm thickness) was heldbetween the top and bottom plates. The circular or rectangular spaceswithin the Teflon spacers on both sides of a membrane were filled withwoven wire meshes which served as membrane support and liquiddistributor. Appropriately positioned screws were used to hold the topand bottom plates together. The effective membrane area in both of thesedevices was 12.57 cm². The effective diameter of membrane used in thecircular module which corresponded to the area of the space within theTeflon spacer was 40 mm whereas the effective length and breadth of therectangular membrane housed within the laterally-fed device was 157 mm×8mm. The dimensions of the inlet and outlet were minimized as much aspossible to reduce the dead volume of these devices. In addition to theinlet and outlet, the modules were provided with additional ports forpriming and removal of bubbles prior to each run.

The dye tracer experiments were performed using ten times diluted foodcolour solutions. The dye was found to bind strongly to the Sartobind Qmembrane and so hydrophilized PVDF membranes having 0.22 μm pore sizewas used in the dye experiments. Degassed microfiltered water was pumpedat a flow rate of 10 mL/min from a reservoir to the membrane modulesusing a HiLoad P-50 pump (GE Healthcare, Piscataway, N.J., USA). Asample injector fitted with a 250 μL loop, installed between the pumpand module was used to introduce the food dye into the devices. Thetransparent (acrylic) side of the membrane module was illuminated usinga table lamp. A digital camera (Sony Cyber-shot, Model DSC-WX7, Japan)was used to take video clips of the membrane surface during the dyeexperiments. Video clips were recorded in MTS format and the extent ofzooming together with the location of the camera relative to the modulewas kept the same in all the experiments. Snapshots were obtained fromthe video files at the rate of one every second using Windows Live MovieMaker and processed using Image J freeware (http://imagej.nih.gov/ij/).The gray scale intensities of the snapshots were measured by codingmacros. For the circular membrane module, intensities of pixels on theradius from the center to the periphery (73 pixels in all) were recordedfor all 30 frames and were multiplied by the area of the circularincrement corresponding to the distance of the pixel from center. Forthe laterally-fed module, intensities of the pixels were measured alongthe length of the membrane (570 pixes in all) and were multiplied by thewidth of the membrane. To avoid any discrepancies owing to theexperiment-to-experiment variations, the intensities were normalized bysubtracting the base line intensity for each pixel, this being theintensity at time zero.

For the protein experiments, the modules fitted with Sartobind Qmembrane were integrated with an AktaPrime liquid chromatography system(GE Healthcare Bioscience, QC, Canada) using PEEK tubings. Phosphatebuffer (20 mM, pH 7.0) was used as the binding buffer as well as forpreparing the feed protein solutions. The eluting buffer consisted tothe binding buffer containing in addition, 0.5 M NaCl. All buffers weredegassed and filtered using PVDF filters (VVLP04700, 0.1 μm pore size,Millipore, Billerica, Mass., USA) just before carrying out the membranechromatography experiments which were carried out at 10 mL/min flowrate. In experiments where lysozyme was used as the unbound tracer,lysozyme solution was injected using a 250 μL sample loop. Breakthroughexperiments were carried out by injecting BSA solutions of appropriateconcentration prepared in the binding buffer. A 50 mL superloop was usedto inject the protein solution into the modules. The BSA bound to themembrane was eluted using buffer containing 0.5 M sodium chloride. Thevoid volume of the membrane modules was determined using lysozyme whichdid not bind to the Sartobind Q membrane and the breakthrough curveswere corrected accordingly. BSA binding experiments were also carriedout in the pulse mode by injecting 100 μL of BSA solution.

FIG. 23 shows snapshots from the first twelve seconds of the dyeexperiments carried out with the stacked-disk and laterally-fed modules.The aim of these dye experiments was to visually compare the flowdistribution on the feed side as well as the effluent collection on thepermeate side of the membrane modules. As already stated, only the topacrylic plates of the modules were transparent. The inlet and outletconnections were therefore swapped around to make either the feed sideor the permeate side transparent. This was feasible as top and bottomplates, though made of different material, were identical in design. Thetime zero in each case was designated to the instant at which the dyejust entered the module. The snapshots of the stacked-disk module showedthe dye breaking through the central region of the module after threeseconds, with the peripheral region of the feed side of the membranestill free of dye. After six seconds, the central region on the feedside started getting depleted of dye while the dye breakthrough in theperipheral region was yet to happen. After twelve seconds, the feed sideof the membrane was largely free of dye except for the periphery and theuniform coloration on the permeate side was due to the dye breakingthrough the peripheral region and migrating to the outlet located at thecenter of the module. Overall, the snapshots obtained with thestacked-disk module clearly indicate acute maldistribution of dye.Therefore the breakthrough binding capacity with this device would beexpected to be low and flow-through and eluate peaks would be broad. Thesnapshots obtained with the device show very good correlation in dyeintensity on the feed and permeate side with excellent lateraldistribution. Therefore the fluid flow and distribution was ashypothesized, i.e. the variability in solute flow path length was low.Based on this, it could be anticipated that the efficiency of separationusing the laterally-fed membrane module would be high.

FIG. 24 represents the quantitative analysis of dye experiments. The 3Dgraphs show the variations of normalized gray scale intensity with timeand location within the two membrane modules. The normalized gray scaleintensity could be considered to be proportional to the amount of dye ata given location. With the stacked-disk membrane module, the peakheights increased from the center to the periphery as the membrane areasincreased in a radially outward direction. The graphs also show that ittook longer for the dye to reach the periphery and once this happened,the dye lingered in the peripheral regions of the membrane for a longtime. On the other hand, the graphs for the laterally-fed membranemodule showed a much more gradual shift of the peak which had a largelyconstant height, from the inlet to the outlet side, clearly indicating afar more uniform solute path length. These results once again predicthigher efficiency of separation with the laterally-fed membrane module.

Tracer experiments were carried out with Sartobind Q membrane usinglysozyme as non-binding protein. FIG. 25 shows the flow-through peaksobtained with the two modules using 250 μL of 2 mg/mL lysozyme solutionsunder non-binding condition (i.e. at pH=7.0). The peak obtained with thelaterally-fed membrane module was significantly sharper and moresymmetrical. With the stacked-disk module, lysozyme appeared in theeffluent earlier, even though the dead volume of the stacked-disk modulewas larger than that of the laterally-fed membrane module. This wasconsistent with the results of the dye experiments discussed earlier,where very early breakthrough at the central region of the membrane wasobserved. The broadening of the peak with the stacked-disk module, whichis indicative of flow maldistribution, was consistent with the dyeexperiments where the dye lingered on in the peripheral regions forquite some time before reaching the outlet. The widths at half heightand the standard deviations of the flow-through peaks obtained with thetwo modules are shown in Table 2. These values indicate that the flowdistribution and thereby the uniformity of solute path length wassignificantly superior with the laterally-fed membrane module.

TABLE 2 Width of Flow-through peaks of a stacked disk device and alaterally fed device. Peak width at Standard half height (mL) deviationStacked-disk 1.386 0.588 Laterally-fed 0.74  0.314

FIG. 26 shows the breakthrough curves for the laterally-fed andstacked-disk membrane modules, obtained by injecting 30 mL of 2 mg/mLBSA solution using a superloop. The void volume correction was madeusing data obtained from experiments carried out using the same volumeof 0.5 mg/mL of lysozyme with the same superloop. The Sartobind Qmembrane had identical bed volumes of 0.346 mL in the two devices. Theincipient and 10% breakthrough BSA binding capacity obtained with thetwo membrane modules are reported in Table 3. These results clearlydemonstrate how the design of the membrane module could have a profoundimpact on the utilization of membrane binding capacity. The quicksaturation of the central regions of the membrane housed in thestacked-disk membrane module by BSA molecules resulted in an earlybreakthrough. By contrast, the breakthrough took place a lot later withthe laterally-fed membrane module, the resultant incipient breakthroughBSA binding capacity being almost five times higher. The 10%breakthrough binding capacity obtained with the laterally-fed membranemodule was 38.46 mg/mL which is an extremely high value for a singlelayer of membrane. The BSA elution peaks obtained in the abovebreakthrough experiments along with the corresponding conductivityprofiles are shown in FIG. 27. The standard deviation and width at halfheight values for the BSA elution peaks obtained with the two modulesare summarized in Table 4. As expected, the laterally-fed membranemodule gave sharper and more symmetrical peak. Moreover, the sharperconductivity profile observed with the laterally-fed membrane moduleindicates its superior design. Broadening of peaks, in addition to beingundesirable in multi-component separation processes, results in productdilution which increases bioseparation cost in large-scale separationprocesses.

TABLE 3 Incipient and 10% breakthrough BSA binding capacity obtainedwith as tacked disk device and a laterally fed device Incipient 10%breakthrough breakthrough binding capacity binding capacity (mg/mL)(mg/mL) Stacked-disk 5.780 8.995 Laterally-fed 28.896 38.461 Ratio 4.9994.276

TABLE 4 standard deviation and width at half height values for the BSAelution peaks obtained with a stacked disk device and a laterally feddevice. Peak width at half Standard height (mL) deviation Stacked-disk2.84 1.206 Laterally-fed 2.05 0.871

FIG. 28 shows the chromatograms obtained in pulse binding experimentscarried out with both modules using 100 μL of 1 mg/ml BSA solution.While there was very little BSA flow-through with the laterally-fedmembrane module, a significant peak, indicating incomplete proteincapture was observed with the stacked-disk module. Table 5 summarizesthe standard deviation and width at half height values for the BSAelution peaks obtained with the two modules. As in the experimentsdescribed in the previous paragraph, the BSA peak was sharper and moresymmetric with the laterally-fed membrane module.

TABLE 5 Standard deviation and width at half height values for the BSAelution peaks obtained with a stacked disk device and a laterally feddevice. Peak width at Standard half height (mL) deviation Stacked-disk2.749 1.167 Laterally fed 1.648 0.699

The above results clearly demonstrate the superiority of thelaterally-fed membrane module over the conventional stacked-disk module.They also highlight the critical role played by the membrane moduledesign on the efficiency of membrane binding capacity utilization. Thelaterally-fed design examined in the current study reduces thevariability in solute path length within the device and thereby leads inmore uniform usage of membrane. In order for the laterally-fed designfeature to be effective, the following conditions have to be met.Firstly, the hydraulic resistance offered support material (woven wiremesh in this case) within which lateral flow distribution and collectiontakes place has to be lower than that offered by the membrane. Secondly,the resistance to lateral flow in the support material on both sides ofthe membrane has to be identical. A higher resistance on the feed sidewould result in greater flow in the membrane closer to the inlet while ahigher resistance on the permeate side would result in greater flowcloser to the outlet. Finally, the aspect (i.e. length to width) ratioof the device is quite important. A low aspect ratio could result inmaldistribution with more lateral flow of feed taking place closer tothe centerline of the support material. A very high aspect ratio on theother hand would result in poor utilization of membrane closer to theoutlet due to increase in lateral resistance.

High-resolution protein purification: head-to-head comparison with aradial-flow device

As the next step, a scaled-up, laterally-fed membrane device housing astack of rectangular membranes was designed and its performance wascompared with equivalent a radial flow membrane device (Sartobind S,Sartorius, Goettingen, Germany) having the same bed volume and bedheight. Tracer experiments using salt were carried out in bothstep-input and pulse mode through which the residence time distributionof the devices were compared. Lysozyme was used for the singlebind-and-elute experiments before conducting protein separations.Finally, ovalbumin, conalbumin, and lysozyme were used as model proteinto run multi-component bind-and-elute experiments.

The prototype device designed for this study is shown in FIG. 1. Thedevice consisted of two acrylic plates 3-D printed using Projet HD3000(3D systems, Rock Hill, S.C., USA) which contained the rectangular feedand permeate channels (70 mm×20 mm). The channels were connected to theinlet and outlet ports through slanted tapered channels. The other twoports were used for priming and removing the bubbles prior to runningexperiments which were kept closed during the chromatography processes.The rectangular channels were filled with a single layer of wire meshhaving approximately the same thickness with their depth (0.5 mm) whichalso helped reducing the dead volume of the system. The middle frame hadthe same outer dimension with the plates and was made with delrin. Thethickness of the frame was equal to the bed height of the stackcontaining a slot with the same dimension of the membranes (70 mm×20 mm)on one side and tapered out on the other side providing enough space forgluing around the membranes. Cation exchange (18 layers) S membranes(Sartorius, Goettingen, Germany) were cut using a metal stamp and gluedwithin the slot using RTV 108 adhesive.

The devices were connected to AKTA prime liquid chromatography system(GE healthcare Biosciences, QC, Canada) using PEEK tubing. The sanitaryconnectors on the radial-flow device were modified using delrin insertswhich decreased the total dead volume of the device. The dead volume ofboth devices was measure with the volume of water required to fill themup. The values were 4.8 mL and 21.0 mL for the LFMC and radial-flowdevices respectively.

The results for the salt tracer (0.5M NaCl) experiments in the pulsemode with 2 mL and 5 mL sample loop are shown in FIG. 29. Phosphatebuffer (20 mM, pH 7) was used as the running buffer. For both pulsevolumes, the conductivity peaks obtained from the LFMC device weresharper and more symmetrical whereas the ones from the radial-flowdevice had significant tailing. The peak width at half height, asymmetryparameter, and tailing factor are shown in Table 6. The results confirmpoor flow distribution within the radial-flow device. Moreover, lookingat the volumes at which the salt appeared in the permeate the value forthe LFMC device being 4.7 mL corresponds very well with the dead volumeof the device. This is while this value for the radial-flow device (8.8mL) is much lower than the device dead volume. The results show severevariability in the flow path length and short-circuiting within theradial-flow device which results in low efficiencies in chromatographicseparation.

TABLE 6 Loop Peak width size at half Asymmetry Tailing FIG. (mL) Deviceheight (mL) parameter factor 29A 2.0 Radial-flow 12.19 3.03 2.33Laterally-fed 3.72 1.33 1.34 29B 5.0 Radial-flow 13.37 3.26 2.37Laterally-fed 6.54 1.29 1.28

FIG. 30 shows the conductivity graphs obtained from both devices in thestep-input mode with 150 mL of salt injection. The salt breakthroughobtained from the radial-flow device (20-120 mL) were shallow andjagged, indicating the poor flow distribution within the device which isalso a result of large dead volume. On the other hand, the saltbreakthrough obtained from the LFMC device was sharp with the incipientbreakthrough matching the dead volume of the system which shows theuniformity in the flow path lengths and close to plug-flow behavior.This could be also inferred from comparing the salt conductivity decayswithin the devices.

The results obtained from the bind-and-elute single protein (8 mg/mLlysozyme) experiments using 2 mL and 5 mL sample volumes are shown inFIGS. 31 and 30, respectively. Sodium phosphate buffer (20 mM, pH 7) wasused as the binding buffer and the eluting buffer was the same buffercontaining 0.5M NaCl. In both sets of experiments run with the LFMCdevice the eluted peaks were significantly sharper and more symmetrical.However, the peaks obtained from the radial flow device were not onlybroadened but also contained shoulder for a single protein whichindicated the non-uniformity of protein binding and elution within theradial-flow device. In comparison, the samples obtained from theradial-flow device were much more diluted which is highly undesirable inlarge-scale separation processes. The peak width at half height valuesfor both sets of experiments are available in Table.7 which clearlydemonstrate the likelihood of excellent multi-component separation usingthe laterally-fed device. Further single protein experiments werecarried out using ovalbumin (pI 4.5), conalbumin (pI 6.1), and lysozyme(pI 11.0) with 2 mL sample loops. Citrate buffer (20 mM, pH 5.5) wasused as the binding buffer and the same buffer containing 0.5M NaCl wasused as the eluting buffer. Based on the pI values of the proteinsovalbumin was expected to flow through while conalbumin and lysozymecould be separated using a linear gradient which confirmed thefeasibility of separating the three proteins in a multi-componentseparation format. FIG. 33 shows the chromatograms obtained from thethree proteins using a 40 mL linear gradient. The flow through ovalbuminpeak was very broad and the eluting peaks were jagged. The resultsindicated that it would not be possible to fully resolve conalbumin andlysozyme with 40 mL gradient with the radial-flow device.

TABLE 7 Loop size Peak width at (mL) Device half height (mL) 2.0Radial-flow 15.81 Laterally-fed 4.21 5.0 Radial-flow 18.00 Laterally-fed6.43

FIG. 34 shows the results obtained from the multi-component separationof the three abovementioned proteins using the radial-flow device. Nearbaseline resolution was achieved with 80 mL gradients. The flow throughpeak was very broad and the eluting peaks were broad and jagged and 25membrane bed volume of buffer was required for the whole separation.

FIG. 35 shows the same results obtained from the LFMC device with threegradients of 20, 30, and 40 mL gradients. The ovalbumin flow throughpeak was much sharper compared to the one acquired from the radial-flowdevice. Therefore, the gradient elution was commenced 5 mL after thesample was injected; the value which was 4 times lower compared to theone required for the radial-flow device. The eluting conalbumin andlysozyme peaks were much sharper and taller and therefore even 30 mLgradient gave almost near baseline resolution. The three model proteinswere fractionated in 10 membrane bed volumes which would make a greatimpact in the large-scale application through drastic decrease in thebuffer consumption, diminishing the processing time, and avoiding sampledilutions. The peaks obtained from the 40 mL gradient experiments werecollected and analyzed with SDS-PAGE. The bands clearly demonstrateexcellent separation of the proteins (see FIG. 33).

The results demonstrate the suitability of the LFMC device forconducting high-resolution, multi-component separations in thebind-and-elute format. The LFMC device offers simple design andfabrication and its flat shape versus the cylindrical shape of theradial-flow devices offer much lower footprints.

While the above description provides examples of one or more methods orsystems, it will be appreciated that other methods or systems may bewithin the scope of the claims as interpreted by one of skill in theart.

What is claimed is:
 1. A method of forming a frame around a membranestack for a laterally-fed membrane chromatography device, the methodcomprising: placing a membrane stack having one or more membrane layerson a bottom surface of body of a master mold, the body having opposedside walls and opposed end walls, the opposed side walls spaced apart bya distance greater than a length of the membrane stack, the opposed endwalls spaced apart by a distance greater than a width of the membranestack; placing a cap on the body of the master mold to enclose themembrane stack in the master mold, the cap having at least one openingfor injecting a material into a space defined by the end walls of themaster mold, the side walls of the master mold, end walls of themembrane stack, side walls of the membrane stack, the bottom surface ofthe body and an inner surface of the cap; injecting the material intothe space around the membrane stack; and curing the material to form aframe around the membrane stack.
 2. The method of claim 1 furthercomprising removing the cap piece and the master mold from the membranestack and frame.
 3. The method of claim 1, wherein the curing thematerial includes cooling the material below a curing temperature. 4.The method of claim 1, wherein the material is a thermoplastic polymerthat is injected into the master mold as a liquid and hardens whencooled.
 5. The method of claim 1, wherein the curing the materialexposing the material to ultraviolet light.
 6. The method of claim 1,wherein the placing the cap on the body of the master mold includesresting the inner surface of the cap on a top surface of the membranestack.
 7. The method of claim 1, wherein the placing the cap on the bodyof the master mold includes resting the inner surface of the cap on anabutment member of the body to support the cap above a top surface ofthe membrane stack.
 8. The method of claim 1, wherein the placing themembrane stack on the bottom surface of the body includes coupling themembrane stack to the bottom surface of the body.
 9. The method of claim1, wherein the placing the membrane stack on the bottom surface of thebody includes placing the membrane stack between retention ridges of thebody to couple the membrane stack to the body.
 10. The method of claim1, wherein the placing the membrane stack on the bottom surface of themaster mold includes positioning the membrane stack on the bottomsurface so the end walls of the membrane stack are adjacent to the endwalls of the body and the side walls of the membrane stack are adjacentto the side walls of the body.
 11. The method of claim 1, wherein duringthe injecting the material into the space around the membrane stack, thematerial is contained within the space around the membrane stack. 12.The method of claim 1, wherein during the curing of the material, thematerial adheres to the side wall and end walls of the membrane stack toform a frame around the membrane stack.
 13. A membrane stack and frameformed by the method of claim
 1. 14. A laterally-fed membranechromatography device comprising a membrane stack and frame formed bythe method of claim 1.